2.1. The Glutamine Synthetase System
In higher plants, many steps in nitrogen metabolism occur in multiple subcellular compartments. For example, isoenzymes for many amino acid biosynthetic enzymes are located in the cytosol, as well as in the mitochondria or chloroplasts. The significance of this multiplicity and compartmentalization of plant isoenzymes has yet to be fully understood. The relative function of many amino acid biosynthetic isoenzymes has been difficult to assess due to inadequate fractionation of organelle and cytoplasm components, overlapping activity profiles, and immunological cross-reactivity (Miflin and Lea, 1982, in "Nucleic Acid and Proteins in Plants I: Structure, Biochemistry and Physiology of Proteins," eds. Boulter, D & Parthier, B, Springer-Verlag, Berlin Heidelberg New York, pp. 5-64). Consequently, it is unclear whether these isoenzymes carry out redundant or distinct roles in plant metabolism.
The best studied example of a plant amino acid biosynthetic enzyme shown to occur as multiple isoforms is glutamine synthetase (GS) (EC 6.3.1.2) (McNally et al., 1983, Plant Physiol. 72:22-25). Early biochemical data revealed that GS functions in the assimilation of ammonia generated by numerous plant processes which include seed germination (Kern and Chrispeels, 1978, Plant Physiol. 62:642-647; Winter et al., 1982, Plant Physiol. 69:41-47), photorespiration (Wallsgrove et al., 1983, Plant Cell Environ. 6: 301-309; Wallsgrove et al., 1987, Plant Physiol. 83:155-158), nitrite reduction (Miflin, 1974, Plant Physiol. 54: 550-555), nitrogen-fixation in root nodules (Robertson et al., 1975, Aust. J. Plant Physiol. 2:265-272; Lara et al., 1983, Plants 157: 254-258), and primary ammonia assimilation from the soil (Hirel and Gadal, 1980, Plant Physiol. 66:619-623). An analysis of the GS genes in several species has revealed a strong correlation of individual GS gene expression with specific aspects of plant development (Tingey et al., 1987, EMBO J. 6:1-9; Tingey et al., 1988, J. Bio. Chem. 263:9651-9657; Hirel et al., 1987, EMBO J. 6:1167-1171; Forde et al., 1989, Plant Cell 1:391-401; Gebhardt et al., 1986, EMBO J. 5:1429-1435; Edwards and Coruzzi, 1989, Plant Cell 1:241-248). Recent sequence analysis of GS cDNAs from Pisum sativum and Phaseolus vulgaris has shown that chloroplast and cytosolic GS are encoded by separate but similar nuclear genes (Tingey et al., 1987, EMBO J. 6:1-9; Tingey et al., 1988, J. Bio. Chem. 263:9651-9657; Cullimore et al., 1984, J. Mol. Appl. Genet. 2:589-599).
In pea, the single nuclear gene for chloroplast GS2 is expressed predominantly in leaves in a light-dependent fashion (Tingey et al., 1988, J. Bio. Chem. 263:9651-9657; Edwards and Coruzzi, 1989, Plant Cell 1:241-248). The role of chloroplast GS2 in the reassimilation of photorespiratory ammonia is supported by the analysis of mutants in barley (Wallsgrove et al., 1987, Plant Physiol. 83:155-158), and is substantiated by gene expression studies in pea (Edwards and Coruzzi, 1989, Plant Cell 1:241-248). For cytosolic GS, molecular studies have revealed the presence of a number of distinct isoforms in several plant species (Tingey et al., 1988 J. Bio. Chem. 263:9651-9657; Hirel et al., 1987, EMBO J. 6: 1167-1171; Gebhardt et al., 1986, EMBO J. 5:1429-1435; Tingey and Coruzzi, 1987, Plant Physiol. 84:366-373). In pea it has been shown that two classes of genes encode homologous but distinct cytosolic GS isoforms (Tingey, 1988, J. Bio. Chem. 263:9651-9657). One class comprises a pair of "twin" GS genes (GS3A and G3B) whose expression is specifically induced in two developmental contexts where large amounts of ammonia are mobilized for plant growth, during germination and nitrogen fixation.